Concentrations of long-chain acyl-acyl carrier proteins during fatty acid synthesis by chloroplasts isolated from pea (Pisum sativum), safflower (Carthamus tinctoris), and amaranthus (Amaranthus lividus) leaves.
Fatty acid synthesis from [1-14C]acetate by chloroplasts isolated from peas and amaranthus was linear for at least 15 min, whereas incorporation of the tracer into long-chain acyl-acyl carrier protein (ACP) did not increase after 2-3 min. When reactions were transferred to the dark after 3-5 min, long-chain acyl-ACPs lost about 90% of their radioactivity and total fatty acids retained all of theirs. Half-lives of the long-chain acyl-ACPs were estimated to be 10-15 s. Concentrations of palmitoyl-, stearoyl-, and oleoyl-ACP as indicated by equilibrium labeling during steady-state fatty acid synthesis, ranged from 0.6-1.1, 0.2-0.7, and 0.4-1.6 microM, respectively, for peas and from 1.6-1.9, 1.3-2.6, and 0.6-1.4 microM, respectively, for amaranthus. These values are based on a chloroplast volume of 47 microliters/mg chlorophyll and varied according to the mode of the incubation. A slow increase in activity of the fatty acid synthetase in safflower chloroplasts resulted in long-chain acyl-ACPs continuing to incorporate labeled acetate for 10 min. Upon re-illumination following a dark break, however, both fatty acid synthetase activity and acyl-ACP concentrations increased very rapidly. Palmitoyl-ACP was present at concentrations up to 2.5 microM in safflower chloroplasts, whereas those of stearoyl- and oleoyl-ACPs were in the lower ranges measured for peas. Acyl-ACPs were routinely separated from extracts of chloroplasts that had been synthesising long-chain fatty acids from labeled acetate by a minor modification of the method of Mancha et al. (Anal. Biochem., 1975, 68, 600-608). The results compared favorably with those obtained using alternative analytical methods such as adsorption to filter paper and partition chromatography on silicic acid columns. The acyl-ACP which coprecipitated with ammonium sulfate was not affected by treatments with neutral hydroxylamine or borohydride, whereas that eluted from silicic acid was relatively easily derivatized. A single radioactive polypeptide of Mr 11,500 from pea and amaranthus chloroplasts was revealed by autoradiography of gels from sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the silicic acid eluates.
Roughan
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Division of Horticulture and Processing, DSIR, Mt. Albert Research Centre, Private Bag, Auckland.
Nishida
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I
D000085:Acetates / Q000378:metabolism; D019342:Acetic Acid; D000213:Acyl Carrier Protein / Q000378:metabolism*; D000409:Alanine / Q000378:metabolism; D002250:Carbon Radioisotopes; D002736:Chloroplasts / Q000378:metabolism*; D003624:Darkness; D007887:Fabaceae / Q000378:metabolism; D005227:Fatty Acids / Q000096:biosynthesis*; D005230:Fatty Acids, Nonesterified / Q000378:metabolism; D007700:Kinetics; D008027:Light; D010944:Plants / Q000378:metabolism*; D010946:Plants, Medicinal; D013045:Species Specificity; D014316:Tritium
D000085:Acetates; D000213:Acyl Carrier Protein; D002250:Carbon Radioisotopes; D005227:Fatty Acids; D005230:Fatty Acids, Nonesterified; D014316:Tritium; D000409:Alanine; D019342:Acetic Acid